The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signaling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behavior that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signaling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signaling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. DAG-lactones represent a synthetically accessible platform for probing the structure activity relations of protein kinase C and the other targets downstream of the second messenger diacylglycerol. In a collaborative effort with the Kazanietz lab, we show that DAG ligands can achieve strong selectivity for PKC epsilon versus PKC alpha and that this selectivity can lead to differential PKC isotype down regulation. In other studies, we further refine our understanding of the features of dimeric DAG-lactones that might exploit the different spacing of the two C1 domains of different PKC isoforms as well as differences in C1 domain selectivity for generating ligands with marked isoform selectivity. Additionally, we have continued to evaluate DAG-lactones designed to have selectivity for the RasGRP subclass of diacylglycerol targets. This class of targets is of particular importance because it functions as an activator of Ras, which is many tumors shows enhanced activity without being mutated. Selectivity is being evaluated both at the in vitro level and in intact cells. Bryostatin is an agent in clinical trials with a unique mechanism of action. It binds to protein kinase C with high affinity and activates the enzyme but paradoxically antagonizes many protein kinase C mediated responses. We find, using microarray analysis of gene expression and aptamer arrays for analysis of protein expression, along with detailed examination of the time and dose dependence of genes representative of the differences in expression revealed by the microarray analysis, that transient duration is the predominant difference in the mode of action of bryostatin as compared to typical protein kinase C activators such as the phorbol esters. In collaboration with the group of Gary Keck, we seek to define the critical structural elements in bryostatin conferring its unique pattern of activity, with the goal of developing the next generation of bryostatin analogues. An important recent advance was to show that subcellular localization of bryostatin analogs cannot explain whether they function as phorbol ester analogs or as phorbol ester antagonists. Further, we show that bryostatin derivatives that only slowly penetrate into cells at no point are able to fully induce the transient responses induced by the phorbol esters. Protein kinase C is subject to post-translational modification, in particular phosphorylation. In collaboration with the CCR Collaborative Protein Technology Resource, we have shown that the extent of protein kinase C modification is much more extensive than had been recognized. Moreover, the pattern of modification was different for different ligands such as phorbol ester or bryostatin.We suggest that such modification signatures may be of particular value for the structure activity evaluation of ligands with complex effects where, as in the case of protein kinase C, ligand binding causes both activation and change in subcellular localization. Comparing the various cell lines of the NCI60 cell line panel, we have shown that the patterns of PKC delta modification show marked differences between the various cell lines. Modification of DAG signaling in these various cell lines causes changes in the patterns of modification, but the changes are much smaller than those associated with phorbol ester, emphasizing that phorbol ester treatment in not a good surrogate for physiological levels of DAG. In addition to those proteins that recognize DAG and phorbol esters through their C1 domains, other proteins contain homologous C1 domains that fail to recognize DAG or phorbol ester. We are actively involved in understanding the structural basis for these differences. Systems examined include the C1 domains of RasGRP2 and Vav2/3 as well as the C1a domain of PKC theta. Additionally, we have probed the role of the various domains of RasGRP1 and RasGRP3 for membrane recognition. Contrary to expectation, we find that the REM domain of RasGRP3 acts as a major negative regulator of its association with the plasma membrane. Moreover, we see little of the predicted influence of the PT domain of RasGRP1 on its regulation by PIP3, which is highly elevated in cells with a constitutively active PI3K or with a functional knockout of PTEN. On our TRPV1 project, we have made considerable progress in defining the optimal structures for binding of antagonists to human TRPV1, as part of our effort which seeks to advance the development of drug candidates for this target. We are exploring the use of in silico screening to define new classes of ligands for TRPV1 and for TRPV2. We have developed a novel ligand with improved characteristics for the screening and evaluation of vanilloids at human TRPV1, solving a problem occasioned by the substantial difference in SAR between the human and rodent orthologues, with the standard ligand for receptor assays proving to be optimal for the rodent, rather than the human, TRPV1.